CN103091299A - Laser differential confocal map microimaging imaging method and device - Google Patents
Laser differential confocal map microimaging imaging method and device Download PDFInfo
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Abstract
The invention belongs to the technical field of optical microimaging and spectral measurement, and relates to a high-spatial resolution laser differential confocal map microimaging imaging method and device. The core thought of the method is that a differential confocal detection technology is merged with a spectrum detection technology together, and a dichroic beam splitting system (13) is used to carry out nondestructive separation on Reyleigh scattering light and Raman scattering light, wherein the Raman scattering light is treated by spectrum detection, the Reyleigh scattering light is treated by geometric position detection, based on the characteristic that the position of a zero crossing point of a differential confocal curve (43) is accurately corresponding to the focus position, spectrum information of the excitation spot focus position is captured through zero crossing point triggering, the high-spatial resolution spectrum detection is realized, and a method and a device capable of realizing the high-spatial resolution spectrum detection on micro zones of samples are formed. The method and the device have the advantages of being accurate in location, high in spatial resolution, high in sensitivity of spectrum detection and controllable in measurement of focal spot size, and the like, and have a wide application prospect in the fields of biomedicine, evidence collection of tribunal, and the like.
Description
Technical field
The invention belongs to optical microphotograph imaging and spectral measurement methods field, relate to a kind of laser differential confocal spectrum micro imaging method and device, the three-dimensional appearance reconstruct and the microscopic spectrum that can be used for all kinds of samples are surveyed.
Technical background
Nineteen ninety G.J.Puppels etc. report the confocal laser Raman spectrum microtechnic that the Raman spectrum Detection Techniques are combined with the confocal laser microtechnic of its invention at the Nature periodical, be the revolution breakthrough of Raman technology.This technology had both been inherited the high-resolution tomography feature of confocal microscopy, can carry out spectral analysis to sample again, therefore can realize the high-resolution chromatography of sample microscopic spectrum is surveyed.This remarkable advantage makes confocal laser Raman spectrum microtechnic take the course of its own in the spectrum test field, and develop the important means into a kind of extremely important sample structure and constituent analysis rapidly, make it to be widely used in the leading basic research of the subjects such as chemistry, biology, medical science, physics, geology, court's evidence obtaining, criminal investigation.
At present, the principle of typical confocal laser Raman spectrum detection instrument as shown in Figure 2, laser along light path successively through after the first condenser, the first pin hole, the 8th condenser, the first beam splitting system, quarter-wave plate, object lens, focus on sample, inspire the Raman diffused light that is loaded with the sample spectra characteristic; Mobile sample makes the Raman diffused light of corresponding sample zones of different again by quarter-wave plate and by the first beam splitting system reflection, enters the first spectrometer through focusing after the 4th condenser, the 4th pin hole, the 5th condenser and carries out spectrographic detection.
The fast development of modern science and technology is had higher requirement to microscopic spectrum detectivity and spatial discrimination detectivity, if will improve spatial resolution, must accurately focus system.In optical detection system, its size is minimum when the measurement focal beam spot is positioned at focus, and excitation light intensity is the strongest, therefore in order to obtain high spatial resolution, the spectrum of the strength of excitation light intensity be must be able to capture, thereby its optimal spatial resolving power and optimum spectrographic detection ability obtained.As shown in Figure 1, near the BB ' zone of existing confocal microscopy laser excitation focus O, all can inspire the Raman spectrum of sample, and can be surveyed by the spectrum investigating system after pin hole.thereby the actual detection position of confocal Raman spectra microtechnic often is in BA and A ' B ' district of out of focus in confocal curves, " microcell " thereby that cause actual detection is much larger than measuring beam focus O place spot size, simultaneously, using Raman spectrum, to carry out confocal location signal to noise ratio (S/N ratio) lower, and further reduce the energy of Raman spectrum due to the effect of the blocking meeting of pin hole, enlarge pinhole size raising spectrum percent of pass and can increase the halfwidth of confocal axial location curve, reduce its bearing accuracy, and the confocal pinhole size in existing confocal Raman system is usually between 150 μ m～200 μ m, pinhole size used is relatively large, also can not well play the effect of focusing.Above-mentioned reason has limited the ability that the confocal Raman spectra microscopic system is surveyed microscopic spectrum, restricted its meticulousr microscopic spectrum test with analyze occasion in application, thereby the Focus accuracy that improves system is the key that improves its spatial resolution.
The fine bundle of the people such as Kimberley F in 1996 optical in " Description and Theory of a Fiber-Optic Confocal and Super-Focal Raman Microspectrometer " replaces the method for the microscopical pin hole of confocal Raman spectra, realize the on-mechanical adjusting of " pin hole " size, it does not reduce the Spectral resolution of system when enlarging " pin hole "; E Kenwood Blvd in 2007 etc. propose to make the fluorescence background of sample measurement reduce approximately 3 orders of magnitude by the picosecond laser that uses 3-4ps in conjunction with corresponding instantaneous exposure technology in " Very efficient fluorescent background suppression in confocal Raman microscopy Department of Physics ", have improved the resolving power of confocal Raman spectra microscopy; N.Everall in 2008 etc. point out to adopt large-numerical aperture (NA=1.4) oil immersion objective in " The Influence of Out-of-Focus Sample Regions on the Surface Specificity of Confocal Raman Microscopy ", azimuthal resolution and the signal to noise ratio (S/N ratio) higher than traditional confocal Raman spectrometer can have been obtained, but this method need to be carried out film-making to sample, can not realize noncontact and nondestructive measurement, limit the range of application of system; M.J.Pelletier in 2009 and Neil J.Everall etc. propose to utilize in " Control of Out-of-Focus Light Intensity in Confocal Raman microscopy using optical preprocessing " and proofread and correct the interference that object lens or structure pupil mask have been eliminated the spectral intensity of out of focus position Raman scattering, improve spectrographic detection efficient, greatly reduced confocal Raman system out of focus Raman spectrum to the impact of its significant depth resolving power.
Above-mentioned research, mainly concentrate on the aspects such as light-source system that the confocal Raman spectra microscopic system relates to, spectrum investigating system, focusing objective len system, spectral information processing, although improved the overall performance of spectroscopic system, but significantly do not improve aspect confocal Raman spectra system space resolution characteristic, the spatial resolution that improves Raman spectrum system is still outstanding issue.
In research fields such as physical chemistry, biomedicine, film and medicines, the chemical composition of analytic sample, space distribution, Surface Physical Chemistry character are obtained the more information of sample with the form of image often, therefore Raman spectrum need to be surveyed to be extended to by the single-node analysis mode sample in certain area coverage is carried out detection analysis, i.e. Raman spectrum imaging.Yet, in order to obtain more accurate, abundanter metrical information, both needed long single-point to excite the Raman spectrum detection time during Raman spectrum imaging, needing that again sample is carried out the multiple spot Raman spectrum surveys, its result certainly will make Raman spectrum imaging need long detection time, often reaches several hours and just can complete imaging.But, be subjected to the impact of environment temperature, vibration, air shake etc. larger in the long-time imaging process of instrument, easily make instrument system produce drift, thereby cause sample location to be detected out of focus; Because existing confocal Raman spectra Detection Techniques do not possess the real-time focal point tracking power, thereby in whole imaging process, can't compensate the defocus error of the detecting position deviation introducing of sample, restrict the raising of confocal Raman spectra imaging technique spatial resolving power.
The confocal Raman spectra Detection Techniques in research fields such as drugs detection, the discriminating of the gem and jade true and false, oil-gas exploration, chemical analysis and archaeologies to its dimensional requirement of surveying focal beam spot difference to some extent, and existing confocal Raman detection technology can't accurately be controlled the size of focused spot size, and its result has also limited the application of confocal Raman spectra imaging technique in each field.
In existing confocal Raman spectra detection instrument, systematic collection to the sample scattering light beam in the Raman diffused light that comprises extremely faint, only have systematic collection to the sample scattering light beam in the Rayleigh light beam that comprises 10
-3～10
-6Doubly, therefore, how to utilize in confocal Raman spectra is surveyed abandon in existing spectrum investigating system be better than Raman diffused light 10
3～10
6Rayleigh light beam is doubly assisted and surveyed is the new way of improving confocal Raman spectra Detection Techniques spatial resolution.
Based on above-mentioned situation, the present invention proposes to abandon in sample scattering light that the existing confocal Raman spectra detection system of differential confocal detection system utilization collects is better than sample Raman diffused light 10
3～10
6Rayleigh light beam doubly carries out detected with high accuracy, itself and spectrum investigating system are organically blended, survey when carrying out spatial positional information and spectral information, to realizing high spatial resolution, measuring differential confocal spectrum imaging and the detection of focused spot size controlled " collection of illustrative plates unification ", and the spectrographic detection of the realizing high spatial resolution micro-field tests problem demanding prompt solution that is present spectrum has extremely important theory and learning value.
the concrete thought of patent of the present invention is: laser differential confocal technology and spectrographic detection technology are organically combined, the differential confocal system utilize systematic collection to sample scattering light in the Rayleigh light beam focus of focal beam spot carried out real-time follow-up and locus survey, spectrum investigating system utilize systematic collection to the scattered light of sample in Raman diffused light carry out spectrographic detection, and then differential confocal detection system signal and Raman spectrum detection system signal are organically blended, thereby the Focus tracking of realizing the laser differential confocal Raman spectrum system is surveyed and the controlled detection of spot size, namely realize the high-space resolution detection of Raman spectrum.
Summary of the invention
To the objective of the invention is to have in order overcoming the deficiency that confocal Raman spectra Detection Techniques spatial resolution is difficult to improve now, to propose a kind of laser differential confocal spectrum microscopy tomography method and apparatus with high spatial resolution.
The objective of the invention is to be achieved through the following technical solutions.
Laser differential confocal spectrum micro imaging method provided by the invention,
a) produce exciting light by excitation beam generation system, through the first beam splitting system, after object lens, focus on sample, and inspire Reyleith scanttering light and be loaded with the Raman diffused light of sample spectral characteristic, the Raman diffused light that inspires and Reyleith scanttering light are by in the systematic collection recovering light path, reflexed to dichroic optical system through after object lens by the first beam splitting system, after the dichroic optical system light splitting, Raman diffused light and Reyleith scanttering light are separated from each other, Reyleith scanttering light is reflected and enters the differential confocal detection system, the Raman diffused light transmission enters spectrum investigating system, utilize accurate corresponding this characteristic in differential confocal curve zero crossing and focal position, accurately catch by triggering zero point the spectral information that excites the hot spot focal position, realize the spectrographic detection of high-space resolution,
B) only the Reyleith scanttering light signal that receives is carried out differential subtracting each other when processing, system can carry out the three dimension scale tomography of high-space resolution; When only the spectral signal of the Raman diffused light that receives being processed, system can carry out spectrographic detection; When simultaneously the signal of the Reyleith scanttering light that receives and Raman diffused light being processed, system can carry out the microcell collection of illustrative plates tomography of high-space resolution, i.e. " the collection of illustrative plates unification " of the high-space resolution of sample geometric position information and spectral information;
C) the focus O of the accurate corresponding object lens in differential confocal curve zero crossing place, can carry out accurate tracking to sample in real time in measuring process focuses, guarantee that sample is in the focal position all the time in whole measuring process, suppress the factors such as environment temperature and vibration to the impact of spectral measurement, thereby improve measuring accuracy;
D) the differential confocal curve zero crossing corresponding object focal point O that measures in place, focused spot size is minimum herein, the zone of surveying is minimum, the out of focus zone of the corresponding object lens in other positions of range of linearity BB ', before burnt or the focused spot size in defocused BB ' zone increase with defocusing amount, utilize this characteristics, the z by the adjustment sample is to defocusing amount, and control the size of focal beam spot according to the Surveying Actual Precision demand, realize controlled to sample search coverage size.
In detection method of the present invention, excitation beam can be light beam: line polarisation, rotatory polarization, radial polarisation light etc.; Can also be the structure light beam that is generated by the pupil filtering technology, itself and the coupling of pupil filtering technology can be compressed the measurement focused spot size, improve system's transverse resolution.
In detection method of the present invention, this system can also survey the scattering spectrums such as fluorescence, Brillouin scattering, Compton scattering light
The invention provides a kind of laser differential confocal spectrum microscopic imaging device, comprise that excitation beam produces system, the first beam splitting system, object lens, 3-D scanning worktable, dichroic optical system, spectrum investigating system, differential confocal detection system and data processing module; Wherein, the first beam splitting system, object lens, 3-D scanning worktable are placed on successively excitation beam along light path and produce system's exit direction, dichroic optical system is positioned at the reflection direction of the first beam splitting system, spectrum investigating system is positioned at the transmission direction of dichroic optical system, the differential confocal detection system is positioned at the reflection direction of dichroic optical system, data processing module is connected with the differential confocal detection system with spectrum investigating system, is used for merging and processes the data that spectrum investigating system and differential confocal detection system collect.
In device of the present invention, spectrum investigating system can be common spectrum investigating system, the 5th detector after comprising the 7th condenser placed successively along light path, be positioned at the second spectrometer of the 7th condenser focal position and be positioned at the second spectrometer is used for the top layer spectrographic detection of sample; It can also be confocal spectrum investigating system, comprise the 4th condenser placed successively along light path, be positioned at the 4th pin hole of the 4th condenser focal position, the 3rd detector after being positioned at the 5th condenser after the 4th pin hole, being positioned at the first spectrometer of the 5th condenser focal position and being positioned at the first spectrometer, improve system signal noise ratio and spatial resolution, and to the chromatography spectrographic detection of sample.
In device of the present invention, excitation beam produces system can also comprise light polarization modulator and iris filter, for generation of polarized light and structure light beam.
In device of the present invention, be used for compression and excite the iris filter of hot spot can be between light polarization modulator and the first beam splitting system, can also be between the first beam splitting system and object lens.
In device of the present invention, excitation beam produces the reflection direction that system can also be placed on the first beam splitting system, dichroic optical system is successively placed on the transmission direction of the first beam splitting system along light path, spectrum investigating system is positioned at the transmission direction of dichroic optical system, the differential confocal detection system is positioned at the reflection direction of dichroic optical system, and data processing module connects differential confocal detection system and spectrum investigating system.
In device of the present invention, can also comprise the 4th beam splitting system and be positioned at the microscopic observation system of the 4th beam splitting system reflection direction, be used for sample and slightly take aim at; Wherein, the 4th beam splitting system can produce between system and the first beam splitting system at excitation beam, can also be between the first beam splitting system and object lens.
In device of the present invention, data processing module comprises for the treatment of the differential subtraction module of positional information with for the data fusion module that merges positional information and spectral information.
Beneficial effect
The present invention contrasts prior art and has following innovative point:
1) utilize the zero crossing and accurate corresponding this characteristic in focal position of differential confocal system axial response curve, accurately catch by triggering zero point the spectral information that excites the hot spot focal position, realize the spectrographic detection of high-space resolution;
2) utilize the dichroic light-dividing device to systematic collection to Reyleith scanttering light and the Raman diffused light that is loaded with sample information carry out light splitting, Reyleith scanttering light enters the differential confocal detection system, Raman diffused light enters the Raman spectrum detection system, realize the utilization fully of luminous energy, what make that faint Raman diffused light can can't harm enters the Raman spectrum detection system, improve the system spectrum detection sensitivity, realize the high-space resolution " collection of illustrative plates unification " of sample geometric position information and spectral information;
3) utilize differential confocal technology to carry out hi-Fix to measuring focal beam spot, and real-time follow-up is carried out in the focusing position, eliminate the environmental impacts such as temperature and vibration, realize regulation and control and make the Raman spectrum system detection all the time accurate corresponding minimum excite the sample spectra in focal beam spot zone, significantly improve the existing microscopical microscopic spectrum detectivity of confocal Raman spectra and geometric position detectivity, namely realize high-space resolution;
4) utilize the characteristic of the corresponding different focused spot size in the differential confocal response curve range of linearity, to the focal beam spot position carrying out accuracy controlling, and then the size of control survey focal beam spot, be convenient to the sample of different testing requirements is tested and analyzed, namely realize measuring focused spot size adjustable;
5) with differential confocal microscopic system and the fusion mutually on 26S Proteasome Structure and Function of Raman spectrum imaging system, both can realize the tomography of sample microcell geometric parameter, can realize the spectrographic detection of sample microcell again, namely realize simultaneously microscale tomography, collection of illustrative plates tomography and three kinds of imaging patterns of spectrum test, and significantly improve antijamming capability, linearity and the defocused property of imaging test system.
The present invention contrasts prior art and has following remarkable advantage:
1) merge differential confocal technology and spectrographic detection technology, utilize the accurate location of differential confocal system focusing, significantly improve the spatial resolution of spectrographic detection;
2) utilize the out of focus zone of differential confocal response curve, the regulation and control focused spot size can satisfy different testing requirements, makes system have versatility;
3) the differential confocal focus triggers Detection Techniques, can significantly suppress non-linear, the sample reflectivity of system and surface tilt etc. to the impact of measurement result, be beneficial to measurement that realizes microtexture high resolution, high anti-jamming capacity, high precision and high chromatography ability etc.
Description of drawings
Fig. 1 is differential confocal and confocal microscopy axial response schematic diagram;
Fig. 2 is confocal Raman spectra formation method schematic diagram;
Fig. 3 is laser differential confocal spectrum micro imaging method schematic diagram;
Fig. 4 is laser differential confocal spectrum microscopic imaging device schematic diagram;
Fig. 5 is the laser differential confocal spectrum microscopic imaging device schematic diagram with non-confocal spectrum investigating system;
Fig. 6 is the laser differential confocal spectrum microscopic imaging device schematic diagram with microscopic function;
Fig. 7 is the reflective laser differential confocal spectrum microscopic imaging device schematic diagram with microscopic function;
Fig. 8 is that the laser differential confocal spectrum micro imaging method is implemented illustration with device;
wherein, the 1-excitation beam produces system, 2-laser instrument, 3-the first condenser, 4-the first pin hole, 5-the 8th condenser, the 6-light polarization modulator, 7-iris filter, 8-the first beam splitting system, the 9-1/4 wave plate, 10-object lens, 11-sample, 12-3-D scanning worktable, 13-dichroic optical system, 14-differential confocal detection system, 15-the second beam splitting system, the 16-second condenser lens, 17-the second pin hole, 18-the first detector, 19-the 3rd condenser, 20-the 3rd pin hole, 21-the second detector, the 22-spectrum investigating system, 23-the 4th condenser, 4-the 4th pin hole, 25-the 5th condenser, 26-the first spectrometer, the 27-entrance slit, 28-plane mirror, 29-the first concave reflection condenser, 30-spectrum grating, 31-the second concave reflection condenser, the 32-exit slit, 33-the 3rd detector, the 34-data processing module, the differential subtraction module of 35-, the 36-data fusion module, the 37-microscopic observation system, 38-Kohler illumination system, the 39-three-beam-splitting system, 40-the 4th beam splitting system, 41-the 6th condenser, 42-the 4th detector, 43-differential confocal curve, the confocal Raman curve of 44-, the 45-confocal curves, 46-the 7th condenser, 47-the second spectrometer, 48-the 5th detector.
Embodiment
The invention will be further described below in conjunction with drawings and Examples.
Basic thought of the present invention is to utilize differential confocal to survey and the combine spectrographic detection of realization " collection of illustrative plates unification " of spectrographic detection.
as shown in Figure 3, excitation beam produces system 1 and produces exciting light, through the first beam splitting system 8, after object lens 10, focus on sample 11, and inspire Reyleith scanttering light and be loaded with the Raman diffused light of sample spectral characteristic, the Raman diffused light that inspires and Reyleith scanttering light are by in the systematic collection recovering light path, reflexed to dichroic optical system 13 through after object lens 10 by the first beam splitting system 8, after dichroic optical system 13 light splitting, Raman diffused light and Reyleith scanttering light are separated from each other, Reyleith scanttering light is reflected and enters differential confocal detection system 14 and carry out position sensing, the Raman diffused light transmission enters spectrum investigating system 22 and carries out spectrographic detection.
As shown in Figure 4, this device comprises that the excitation beam of placing successively along light path produces system 1, the first beam splitting system 8, object lens 10, sample 11,3-D scanning worktable 12, be positioned at the dichroic optical system 13 of the first beam splitting system 8 reflection directions, be positioned at the differential confocal detection system 14 of spectrum investigating system 22 and the reflection direction of dichroic optical system 13 transmission direction, also comprise the data processing module 34 that connects spectrum investigating system 22 and differential confocal detection system 14.
Spectrum investigating system in Fig. 4 22 is replaced with the normal optical spectra system that comprises the 7th condenser 46, the second spectrometer 47 and the 5th detector 48, namely pie graph 5.
Add the 4th beam splitting system 40, the four beam splitting system 40 reflection directions between the first beam splitting system 8 and object lens 10 and add microscopic observation system 37 in Fig. 4, namely pie graph 6.
Excitation beam in Fig. 6 is produced the reflection direction that system 1 is positioned over the first beam splitting system 8, and dichroic optical system 13 is positioned over the transmission direction of the first beam splitting system 8, and namely pie graph 7.
Embodiment
In the present embodiment, light polarization modulator 6 is the radial polarisation photogenerator, the first beam splitting system 8 is for protecting inclined to one side Amici prism, the second beam splitting system 15 is for protecting inclined to one side Amici prism, three-beam-splitting system 39 is the broadband Amici prism, the 4th beam splitting system 40 is for protecting inclined to one side Amici prism, and dichroic optical system 13 is Notch filter, and spectrum investigating system 22 is the Raman spectrum detection system.
As shown in Figure 8, the laser differential confocal spectrum micro imaging method, its testing procedure is as follows:
At first, Kohler illumination system 38 produces equal white light, after white light sees through broadband Amici prism 39, protected inclined to one side Amici prism 40 reflections, focus on sample 11 through object lens 10, white light is reflected back toward original optical path, after being reflected respectively by the inclined to one side Amici prism 40 of guarantor, broadband Amici prism 39 after object lens 10, through entering the 4th detector 42 after the 6th condenser 41, by the image of observing in the 4th detector 42, test sample product 11 are slightly taken aim at, needed the zone of observation to carry out coarse positioning to sample 11 to determine sample 11.
then, the light beam that laser instrument 2 sends is through the first condenser 3, the first pin hole 4, after the 8th condenser, 5 collimator and extenders are directional light, become radial polarisation light after light beam process radial polarisation photogenerator 6, radial polarisation light light beam after iris filter 7 is modulated, after seeing through the inclined to one side Amici prism 8 of guarantor, forming the compression hot spot by object lens 10 focuses on sample 11, and inspire Reyleith scanttering light and be loaded with the Raman diffused light of sample 11 spectral characteristics, sample 11 can be processed by strengthening the Raman enhancing technology such as Raman spectrum nano particle, to improve the Raman scattering light intensity.
mobile sample 11, make the Raman diffused light of Reyleith scanttering light and corresponding sample 11 zoness of different be returned original optical path by systematic collection, after also the inclined to one side Amici prism 40 of guarantor is crossed in transmission through object lens 10, protected inclined to one side Amici prism 8 reflections and entered probe portion, wherein, the Raman diffused light transmission is crossed Notch filter13 and is entered Raman spectrum detection system 22, Raman spectrum detection system 22 is the confocal Raman spectra detection system, Raman diffused light is converged to the 4th pin hole 24 by the 4th condenser 23, assemble through the 5th condenser 25 and enter the first spectrometer 26, Raman diffused light is through entrance slit 27, arrive spectrum grating 30 after plane mirror 28 and the first concave reflection condenser 29 reflections, after light beam process spectrum grating 30 diffraction, by the second concave reflection condenser 31 reflect focalizations to exit slit 32, incide at last the 3rd detector 33.Due to the grating diffration effect, in Raman spectrum, different wave length is separated from each other, be monochromatic light from exit slit 32 light out, when spectrum grating 30 rotates, different from the optical wavelength of exit slit 32 outgoing, the response by monitoring the 3rd detector 33 and the angle of grating rotating can obtain the Raman spectrum of sample 11; Reyleith scanttering light is entered differential confocal detection system 14 by Notch filter13 reflection, Reyleith scanttering light through protecting inclined to one side Amici prism 15 transmissions is divided into two bundles, Reyleith scanttering light through protecting 15 reflections of inclined to one side Amici prism is focused on by second condenser lens 16, and to enter apart from second condenser lens 16 focus front distances be that the second pin hole 17 of M position is rear is received by the first detector 18; Reyleith scanttering light through protecting inclined to one side Amici prism 15 transmissions is focused on by the 3rd condenser 19, enters that distance be the 3rd pin hole 20 of M after the 3rd condenser 19 focuses, then by the second detector 21 receptions after the 3rd pin hole 20.
In measuring process, sample 11 is carried out axially and during transversal scanning, two the second detectors 21 and the first detector 18 in differential confocal detection system 14, recording respectively the intensity response of reacting sample 11 concavo-convex variations is I
1(ν, u ,+u
M) and I
2(ν, u ,-u
M), with gained intensity response I
1(ν, u ,+u
M) and I
2(ν, u ,-u
M) be sent to differential subtraction module 35 and carry out the differential processing of subtracting each other, obtain differential confocal intensity response I (ν, u, u
M):
I(ν，u，u
M)＝I
1(ν，u，+u
M)-I
2(ν，u，-u
M) (1)
Thereby realize the microscopy tomography of sample 11 geometric positions, in formula (1), v is horizontal normalization optical coordinate, and u is axial normalization optical coordinate, u
MDefocusing amount for pin hole;
The Raman diffused light spectral signal that is loaded with sample 11 spectral informations that in Raman spectrum detection system 22, the 3rd detector 33 detects is I (λ) (λ is wavelength).
With I (λ), I (ν, u, u
M) be sent to data fusion module 36 and carry out data and process, thereby obtain to comprise sample 11 positional information I (ν, u, u
M) and the four-dimensional metrical information I (ν, u, λ) of spectral information I (λ).
Along x, y to scanning, object lens 10 repeat above-mentioned steps along z to scanning to sample 11, record near homologue mirror foci individual positional information I (ν, u, the u of comprising of one group of i in position
M) and the sequence measuring information { I of spectral information I (λ)
i(λ), I
i(ν, u) };
Utilize distinguishable regional δ
iCorresponding positional information I
i(ν, u, uM) finds out corresponding δ
iThe spectral information I in zone
i(λ) value, then according to the relation of v and lateral attitude coordinate (x, y) and the relation of u and axial location coordinate z, reconstruct reflection measured object microcell δ
iThe information I of three dimension scale and spectral characteristic
i(x
i, y
i, z
i, λ
i);
Corresponding minimum distinguishable regional δ
minThree dimension scale and spectral characteristic can be determined by formula (2):
Can realize like this nanoscale microcell laser differential confocal spectrum micro-imaging.
Simultaneously, can utilize the different measuring value { z of differential confocal axial response curve BB ' section
i, determine the spectral characteristic I of corresponding different measuring value position
δ i(z
i, λ
i), can realize exciting near the spectral characteristic test of the controlled microcell of focus.
As can be seen from Figure 8, by the actual zero point O of differential confocal detection system 14, can accurately catch the focal position that excites hot spot, from measuring sequence data { I
i(λ), I
i(ν, u)) } in, the excitation spectrum of extraction corresponding focus positions O has namely been realized microcell δ
minSpectrographic detection and three-dimensional geometry position sensing.
By to metrical information { I
i(λ), I
i(ν, u) } fusion treatment, can realize three kinds of measurement patterns shown in formula (3), that is: microcell collection of illustrative plates tomography test, three dimension scale tomography and spectrum test.
as shown in Figure 8, the laser differential confocal spectrum microscopic imaging device comprises that the excitation beam of placing successively along light path produces system 1, be positioned at the inclined to one side Amici prism 8 of guarantor that excitation beam produces system's 1 exit direction, object lens 10, sample 11, 3-D scanning worktable 12 and be positioned at the Notch filter13 that protects inclined to one side Amici prism 8 reflection directions, be positioned at the Raman spectrum detection system 22 of Notch filter13 transmission direction, be positioned at the differential confocal detection system 14 of Notch filter13 reflection direction and be positioned at the data processing module 34 of differential confocal detection system 14 and Raman spectrum detection system 22 junctions, wherein, excitation beam produces system 1 for generation of excitation beam, comprises along light path placing successively laser instrument 2, the first condenser 3, being positioned at the first pin hole 4, the 8th condenser 5, radial polarisation photogenerator 6 and the iris filter 7 of the first condenser 3 focal positions, the Raman spectrum detection system comprises the 4th condenser 23 placed successively along light path, be positioned at the 4th pin hole 24 of the 4th condenser 23 focal positions, the 3rd detector 33 after being positioned at the 5th condenser 25 after the 4th pin hole 24, being positioned at the first spectrometer 26 of the 5th condenser 25 focal positions and being positioned at the first spectrometer 26, wherein, the first spectrometer 26 comprises entrance slit 27, plane mirror 28, the first concave reflection condenser 29, spectrum grating 30, the second concave reflection condenser 31 and the exit slit 32 of placing successively along light path, differential confocal detection system 14 comprises the 3rd condenser 19, the 3rd pin hole 20, the second detector 21 of protecting inclined to one side Amici prism 15, being positioned at inclined to one side Amici prism 15 transmission direction of guarantor, second condenser lens 16, the second pin hole 17, the first detector 18 that is positioned at inclined to one side Amici prism 15 transmission direction of guarantor, wherein, it is defocused apart from the M place that the 3rd pin hole 20 is positioned at the 3rd condenser 19, and the second pin hole 17 is positioned at the burnt front distance M of second condenser lens 16 place, data processing module 34 comprises differential subtraction module 35 and data fusion module 36, is used for the data that fusion treatment collects.
Below by reference to the accompanying drawings the specific embodiment of the present invention is described; but these explanations can not be understood to limit scope of the present invention; protection scope of the present invention is limited by the claims of enclosing, and any change of carrying out on claim of the present invention basis is all protection scope of the present invention.
Claims (10)
1. laser differential confocal spectrum micro imaging method is characterized in that:
a) produce system (1) by excitation beam and produce exciting light, through the first beam splitting system (8), after object lens (10), focus on sample (11), and inspire Reyleith scanttering light and be loaded with the Raman diffused light of sample (11) spectral characteristic, the Raman diffused light that inspires and Reyleith scanttering light are by in the systematic collection recovering light path, through being reflexed to dichroic optical system (13) by the first beam splitting system (8) after object lens (10), after dichroic optical system (13) light splitting, Raman diffused light and Reyleith scanttering light are separated from each other, Reyleith scanttering light is reflected and enters differential confocal detection system (14), the Raman diffused light transmission enters spectrum investigating system (22), utilize accurate corresponding this characteristic in differential confocal curve (43) zero crossing and focal position, accurately catch by triggering zero point the spectral information that excites the hot spot focal position, realize the spectrographic detection of high-space resolution,
B) only the Reyleith scanttering light signal that receives is carried out differential subtracting each other when processing, system can carry out the three dimension scale tomography of high-space resolution; When only the spectral signal of the Raman diffused light that receives being processed, system can carry out spectrographic detection; When simultaneously the signal of the Reyleith scanttering light that receives and Raman diffused light being processed, system can carry out the microcell collection of illustrative plates tomography of high-space resolution, i.e. " the collection of illustrative plates unification " of the high-space resolution of sample geometric position information and spectral information;
C) the focus O of differential confocal curve (43) the accurate corresponding object lens in zero crossing place (10), can carry out accurate tracking to sample (11) in real time in measuring process focuses, guarantee that sample (11) is in the focal position all the time in whole measuring process, suppress the factors such as environment temperature and vibration to the impact of spectral measurement, thereby improve measuring accuracy;
D) differential confocal curve (43) corresponding object lens (10) the focus O that measures in zero crossing place, focused spot size is minimum herein, the zone of surveying is minimum, the out of focus zone of the range of linearity BB' corresponding object lens in other positions (10), before burnt or the focused spot size in defocused BB' zone increase with defocusing amount, utilize this characteristics, z by adjusting sample is to defocusing amount, and control the size of focal beam spot according to the Surveying Actual Precision demand, realize controlled to sample search coverage size.
2. laser differential confocal spectrum micro imaging method described according to right 1, it is characterized in that: excitation beam is light beam: line polarisation, rotatory polarization, radial polarisation light; Or the structure light beam that is generated by the pupil filtering technology, its and the coupling of pupil filtering technology can be compressed the measurement focused spot size, raising system transverse resolution.
3. laser differential confocal spectrum micro imaging method described according to right 1, it is characterized in that: this system can also survey the scattering spectrums such as fluorescence, Brillouin scattering, Compton scattering light.
4. the laser differential confocal spectrum microscopic imaging device, is characterized in that: comprise that excitation beam produces system (1), the first beam splitting system (8), object lens (10), 3-D scanning worktable (12), dichroic optical system (13), spectrum investigating system (22), differential confocal detection system (14) and data processing module (34), wherein, the first beam splitting system (8), object lens (10), 3-D scanning worktable (12) is placed on successively excitation beam along light path and produces system (1) exit direction, dichroic optical system (13) is positioned at the reflection direction of the first beam splitting system (8), spectrum investigating system (22) is positioned at the transmission direction of dichroic optical system (13), differential confocal detection system (14) is positioned at the reflection direction of dichroic optical system (13), data processing module (34) is connected 14 with spectrum investigating system (22) with the differential confocal detection system) be connected, be used for merging and process the data that spectrum investigating system (22) and differential confocal detection system (14) collect.
5. laser differential confocal spectrum microscopic imaging device described according to right 4, it is characterized in that: spectrum investigating system (22) is common spectrum investigating system, the 5th detector (48) after comprising the 7th condenser (46) placed successively along light path, be positioned at second spectrometer (47) of the 7th condenser (46) focal position and be positioned at the second spectrometer (47) is used for the top layer spectrographic detection of sample; Or confocal spectrum investigating system, the 3rd detector (33) after comprising the 4th condenser (23), the 4th pin hole (24) that is positioned at the 4th condenser (23) focal position placed successively along light path, be positioned at the 5th condenser (25) after the 4th pin hole (24), be positioned at first spectrometer (26) of the 5th condenser (25) focal position and be positioned at the first spectrometer (26), improve system signal noise ratio and spatial resolution, and to the chromatography spectrographic detection of sample.
6. laser differential confocal spectrum microscopic imaging device described according to right 4 is characterized in that: excitation beam produces system (1) can also comprise light polarization modulator (6) and iris filter (7), for generation of polarized light and structure light beam.
7. laser differential confocal spectrum microscopic imaging device described according to right 6, it is characterized in that: be used for compression and excite the iris filter (7) of hot spot can be positioned between light polarization modulator (6) and the first beam splitting system (8), can also be positioned between the first beam splitting system (8) and object lens (10).
8. laser differential confocal spectrum microscopic imaging device described according to right 4, it is characterized in that: excitation beam produces the reflection direction that system (1) can also be placed on the first beam splitting system (8), dichroic optical system (13) is successively placed on the transmission direction of the first beam splitting system (8) along light path, spectrum investigating system (22) is positioned at the transmission direction of dichroic optical system (13), differential confocal detection system (14) is positioned at the reflection direction of dichroic optical system (13), data processing module (34) connects differential confocal detection system (14) and spectrum investigating system (22).
9. laser differential confocal spectrum microscopic imaging device described according to right 4, it is characterized in that: can also comprise the 4th beam splitting system (40) and be positioned at the microscopic observation system (37) of the 4th beam splitting system (40) reflection direction, be used for sample and slightly take aim at; Wherein, the 4th beam splitting system (40) can be positioned at excitation beam and produce between system (1) and the first beam splitting system (8), can also be positioned between the first beam splitting system (8) and object lens (10).
10. laser differential confocal spectrum microscopic imaging device described according to right 4 is characterized in that: data processing module (34) comprises for the treatment of the differential subtraction module (35) of positional information and is used for merging the data fusion module (36) of positional information and spectral information.
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| US14/366,266 US9410880B2 (en) | 2013-01-21 | 2013-08-08 | Laser differential confocal mapping-spectrum microscopic imaging method and device |
| PCT/CN2013/081066 WO2014110900A1 (en) | 2013-01-21 | 2013-08-08 | Method and apparatus for laser differential confocal spectrum microscopy |
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| WO2014110900A1 (en) | 2014-07-24 |
| EP2799844A4 (en) | 2015-09-30 |
| CN103091299B (en) | 2015-01-21 |
| US20150346101A1 (en) | 2015-12-03 |
| EP2799844A1 (en) | 2014-11-05 |
| US9410880B2 (en) | 2016-08-09 |
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